In this competitive renewal, the Proteomics Core is focused on the application and continued improvement of cutting-edge proteomic technologies to study the process of hepatitis C virus (HCV) infection and its impact on liver function, AIDS, and the intravenous drug abuse population. To support these activities, the Center has teamed with the Biological Systems Analysis and Mass Spectrometry group headed by Dr. Richard D. Smith at Pacific Northwest National Laboratory (PNNL). This group brings to the effort unrivaled mass spectrometry instrumentation and world-class proteomics expertise. In Specific Aim 1, the Center will expand upon its use of unique, highly sensitive Fourier transform ion cyclotron mass spectrometry (FTICR-MS) instrumentation available at PNNL to investigate the clinical significance of HCV-associated protein abundance changes using biopsy samples that provide limited protein yields. Applying proteomics to larger sample sets from a broader spectrum of HCV-infected individuals (e.g., intravenous drug abuser and HIV-1 co-infected patients) will provide a better understanding of the host response to HCV and HIV-1 infection and the impact of drug abuse on basic HCV (HIV-1) infection biology. In Specific Aim 2, we will leverage increases in PNNL's throughput capabilities to extend our quantitative proteomic studies to multiple HCV and HIV-1 model systems (animal and cell culture). We will further leverage recent technological and methodological advances that now permit in-depth proteome coverage of high dynamic range samples to analyze additional sample types (human plasma samples complementary to liver biopsy tissues and HCV/HIV cell culture secreted proteins) not proposed in the original grant. The data generated from these studies will suport efforts to provide a detailed molecular portrait of the protein abundance changes that occur during the progression from HCV infection to end-stage liver disease and are likely to yield improved diagnostic methods, markers of disease progression, and novel approaches to therapeutic intervention. In Specific Aim 3, PNNL will provide the informatics resources needed to supply processed peptide identification and quantification data to the NIDA Center for higher-order analyses and biological interpretation. PNNL will also provide the raw MS data in various forms supporting release to the scientific community for independent verification and comparison with other large-scale proteomic datasets.